Pyrano [3,2-C] pyridones and related heterocyclic compounds as pharmaceutical agents for treating disorders responsive to apoptosis, antiproliferation or vascular disruption, and the use thereof

ABSTRACT

A pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier and a compound Formula I: 
                         
or a pharmaceutically acceptable salt or prodrug thereof.

The present invention is related pharmaceutical compounds based on pyrano[3,2-c]pyridine and related fused heterocyclic scaffolds, as represented in Formula I, that are capable of treating a disorder responsive to the induction of apoptosis or antiproliferation or vascular disruption in an animal suffering from such a disorder. The present invention thus relates to the administration to an animal in need of such treatment of an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof, as well as to a pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier and a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof, as well as to a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof.

The compounds of the present application can, for example, induce apoptosis in cancer cells without having a similar effect on non-cancerous cells. Applications for the compounds of Formula I include antiproliferation and vascular disruption agents anticancer drugs for a number of other disorders.

Due to the drug resistance of tumor cells that has developed to known anti-cancer drugs, as well as the severe side effects associated with the use of these known drugs, novel agents having an improved effectiveness and a reduced toxicity are needed. The compounds of the present application inhibit cell division by interfering with microtubule assembly to induce apoptosis (programmed cell death) in cancer cells. The compounds of the present application also interfere with microtubule assembly as vascular disrupting agents (inhibitors of growth of new blood cells). For example, necrosis can be induced in solid tumors by depriving them of oxygen and nutrients.

BRIEF DESCRIPTION OF THE DRAWINGS

To assist in explaining the present invention, the following drawings are provided, in which:

FIG. 1 shows induction of apoptosis in Jurkat cells,

FIG. 2 shows DNA laddering in Jurkat cells,

FIG. 3 shows light microscopy pictures, and

FIG. 4 shows the effect of Applicant's compounds on in vitro tubulin polymerization.

General procedure for the synthesis of pyrano-[3,2-c]-pyridones and related fused heterocycles: A mixture of a required 4-hydroxypyridin-2(1H)-one (0.8 mmol), malononitrile (0.8 mmol), triethylamine (0.05 mL) and a corresponding aldehyde (0.8 mmol) in EtOH (96% aqueous solution, 3 mL) was refluxed for 50 minutes. The reaction mixture was allowed to cool to room temperature, the precipitated product was collected by filtration and washed with EtOH (5 mL). In most cases the product was >98% pure as judged by ¹H NMR analysis. When necessary the products were recrystallized from DMF.

2-amino-5-oxo-4-(3,4,5-trimethoxyphenyl)-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (1), (R═H, X═Ar, Q═CN, Y=aromatic ring, Z═H): 89%; ¹H NMR (DMSO-d₆) δ 11.76 (s, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.57 (m, 1H), 7.34-7.27 (m, 2H), 7.24 (s, 2H), 6.48 (s, 2H), 4.48 (s, 1H), 3.68 (s, 6H), 3.61 (s, 3H).

2-amino-4-(3-bromophenyl)-6-methyl-3-(phenylsulfonyl)-4,6-dihydro-5H-pyrano[3,2-c]quinolin-5-one (2), (R═Alk, X═Ar, Q═PhSO₂, Y=aromatic ring, Z═H): 92%; 8.10 (d, J=8.0 Hz, 1H), 7.67-7.14 (m, 14H), 4.76 (s, 1H), 3.50 (s, 3H).

2-Amino-4-(5-bromo-3-pyridinyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (3), (R═Alk, X═HetAr, Q═CN, Y=aromatic ring, Z =H): 85%; ¹H NMR (DMSO-d₆) δ 8.53 (d, J=2.2 Hz, 1H), 8.50 (d, J=1.6 Hz, 1H), 8.07 (d, J=8.0 Hz, 1H), 7.84 (m, 1H), 7.74-7.69 (m, 1H), 7.56 (d, J=7.6 Hz, 1H), 7.41-7.36 (m, 1H), 7.05 (s, 2H), 4.68 (s, 1H), 3.57 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.7, 159.8, 151.3, 149.2, 145.8, 142.4, 139.7, 132.4, 122.8, 115.5, 108.0, 58.2, 36.0, 29.8; HRMS m/z (ESI) calcd for C₁₉H₁₃BrN₄O₂ (M+Na⁺) 431.0120, found 431.0139.

4-(2-Amino-3-cyano-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinolin-4-yl)-2-bromo-6-methoxyphenyl acetate (4), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 84%; ¹H NMR (DMSO-d₆) δ8.04 (d, J=8.0 Hz, 1H), 7.74-7.69 (m, 1H), 7.57 (d, J=7.6 Hz, 1H), 7.42-7.37 (m, 1H), 7.25 (s, 2H), 7.03 (s, 1H), 7.00 (s, 1H), 4.61 (s, 1H), 3.76 (s, 3H), 3.57 (s, 3H), 2.29 (s, 3H); ¹³C NMR (DMSO-d₆) δ 168.3, 160.5, 159.4, 153.2, 149.6, 142.3, 139.5, 132.8, 122.5, 122.0, 121.7, 120.4, 115.5, 108.1, 57.6, 36.8, 29.6, 20.4; HRMS m/z (ESI) calcd for C₂₃H₁₈BrN₃O₅ (M+Na⁺) 518.0328, found 518.0334.

2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-6-methyl-9-nitro-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (5), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═NO₂): 87%; ¹H NMR (DMSO-d₆) δ 8.89 (s, 1H), 8.50 (d, J=8.1 Hz, 1H), 7.77 (d, J=8.1 Hz, 1H), 7.39 (s, 2H), 7.06 (s, 1H), 6.67 (s, 1H), 4.95 (s, 1H), 3.75 (s, 3H), 3.61 (s, 3H), 3.58 (s, 3H).

2-amino-6-methyl-9-nitro-5-oxo-4-(3,4,5-trimethoxyphenyl)-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (6), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═NO₂): 89%; ¹H NMR (DMSO-d₆) δ 8.88 (s, 1H), 8.47 (d, J=8.0 Hz, 1H), 7.77 (d, J=8.0 Hz, 1H), 7.43 (s, 2H), 6.50 (s, 1H), 4.52 (s, 1H), 3.70 (s, 6H), 3.61 (s, 6H).

2-amino-9-bromo-4-(3-bromo-4,5-dimethoxyphenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (7), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═Br): 92%, ¹H NMR (DMSO-d₆) δ 8.18 (s, 1H), 7.86 (d, J=8.2 Hz, 1H), 7.53 (d, J=8.2 Hz, 1H), 7.25 (s, 2H), 7.05 (s, 1H), 6.64 (s, 1H), 4.93 (s, 1H), 3.75 (s, 3H), 3.60 (s, 3H), 3.50 (s, 3H).

2-Amino-4-[3-bromo-4-(dimethylamino)phenyl]-6,7-dimethyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]pyridine-3-carbonitrile (8), (R═Alk, X═Ar, Q═CN, Y=no ring): 83%; ¹H NMR (DMSO-d₆) δ 7.27-7.08 (m, 5H), 6.06 (s, 1H), 4.28 (s, 1H), 3.33 (s, 3H), 2.66 (s, 6H), 2.33 (s, 3H); ¹³C NMR (DMSO-d₆) δ 161.7, 159.8, 155.4, 153.7, 148.8, 145.0, 143.1, 122.9, 112.7, 105.5, 97.4, 60.5, 56.6, 39.3, 37.2, 31.2, 20.8; HRMS m/z (ESI) calcd for C₁₉H₁₉BrN₄O₂ (M+Na⁺) 437.0589, found 437.0580.

2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-6,7-dimethyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]pyridine-3-carbonitrile (9), (R═Alk, X═Ar, Q═CN, Y=no ring): 87%; ¹H NMR (DMSO-d₆) δ6.93 (d, J=6.9 Hz, 2H), 6.87-6.85 (m, 2H), 6.03 (s, 1H), 4.37 (s, 1H), 3.80 (s, 3H), 3.73 (s, 3H), 3.35 (s, 3H), 2.35 (s, 3H); ¹³C NMR (DMSO-d₆) δ161.7, 159.8, 155.4, 153.6, 148.5, 145.2, 142.7, 123.2, 120.1, 116.8, 113.1, 105.6, 97.4, 60.6, 58.2, 56.8, 37.3, 31.2, 20.6; HRMS m/z (ESI) calcd for C₁₉H₁₈BrN₃O₄ (M+Na⁺) 454.0378, found 454.0371.

2-Amino-4-(3-bromo-4-ethoxy-5-methoxyphenyl)-6,7-dimethyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]pyridine-3-carbonitrile (10), (R═Alk, X═Ar, Q═CN, Y=no ring): 88%; ¹H NMR (DMSO-d₆) δ 7.09 (s, 2H), 6.90 (s, 1H), 6.79 (s, 1H), 6.07 (d, J=2.7 Hz, 1H), 4.33 (d, J=3.0 Hz, 1H), 3.92 (q, J=3.0 Hz, 2H), 3.76 (s, 3H), 3.33 (s, 3H), 2.33 (s, 3H), 1.27 (t, J=3.0 Hz. 3H); ¹³C NMR (DMSO-d₆) δ 161.6, 159.8, 155.4, 153.8, 148.6, 144.3, 142.7, 122.9, 117.5, 112.6, 105.5, 97.42, 68.9, 57.8, 56.6, 37.1, 31.1, 20.8, 16.1; HRMS m/z (ESI) calcd for C₂₀H₂₀BrN₃O₄ (M+Na⁺) 468.0535, found 468.0540.

2-Amino-4-(3-bromo-4-hydroxy-5-methoxyphenyl)-6,7-dimethyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]pyridine-3-carbonitrile (11), (R═Alk, X═Ar, Q═CN, Y=no ring): 75%; ¹H NMR (DMSO-d₆) δ 9.32 (s, 1H), 7.04 (s, 2H), 6.82-6.71 (m, 2H), 6.06 (s, 1H), 4.28 (s, 1H), 3.77 (s, 3H), 3.32 (s, 3H), 2.34 (s, 3H); ¹³C NMR (DMSO-d₆) δ161.6, 159.6, 155.1, 148.5, 148.4, 143.0, 137.5, 123.1, 111.5, 109.6, 105.9, 97.4, 58.0, 56.7, 37.0, 31.1, 20.8;□ HRMS m/z (ESI) calcd for C₁₈H₁₆BrN₃O₄ (M+Na⁺) 440.0222, found 440.0223.

4-(2-Amino-3-cyano-6,7-dimethyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]pyridin-4-yl)-2-bromo-6-methoxyphenyl acetate (12), (R═Alk, X═Ar, Q═CN, Y=no ring): 83%; ¹H NMR (DMSO-d₆) δ7.02 (s, 1H), 6.97 (s, 2H), 6.92 (s, 1H) 6.05 (s, 1H), 4.44 (s, 1H), 3.77 (s, 3H), 3.34 (s, 3H), 2.34 (s, 3H), 2.30 (s, 3H); ¹³C NMR (DMSO-d₆) δ 168.2, 161.9, 159.9, 155.6, 152.5, 148.9, 145.4, 136.5.0, 122.9, 120.2, 116.6, 112.5, 105.4, 97.4, 57.6, 56.7, 37.2, 31.1, 20.5; HRMS m/z (ESI) calcd for C₂₀H₁₈BrN₃O₅ (M+Na⁺) 482.0328, found 482.0322.

2-Amino-4-(3-bromo-4-fluorophenyl)-6,7-dimethyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]pyridine-3-carbonitrile (13), (R═Alk, X═Ar, Q═CN, Y=no ring): 84%; ¹H NMR (DMSO-d₆) δ7.42 (m, 1H), 7.29-7.15 (m, 2H), 7.07 (s, 2H) 6.05 (s, 1H), 4.37 (s, 1H), 2.33 (s, 3H), 2.30 (s, 3H); ¹³C NMR (DMSO-d₆) δ161.6, 159.8, 155.35, 148.7, 143.5, 132.9, 129.3, 120.2, 117.2, 116.8, 108.1, 105.3, 97.5, 57.9, 36.8, 30.9, 20.7; HRMS m/z (ESI) calcd for C₁₇H₁₃BrFN₃O₂ (M+Na⁺) 412.0073, found 412.0073.

2-Amino-4-(3-bromophenyl)-6,7-dimethyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]pyridine-3-carbonitrile (14), (R═Alk, X═Ar, Q═CN, Y=no ring): 97%; ¹H NMR (DMSO-d₆) δ7.39-7.11 (m, 6H), 6.07 (s, 1H), 4.35 (s, 1H), 3.30 (s, 3H), 2.34 (s, 3H); ¹³C NMR (DMSO-d₆) δ 161.5, 159.6, 155.3, 148.7, 148.1, 131.0, 130.7, 129.9, 127.2, 121.9, 120.3, 105.3, 97.4, 57.6, 37.4, 31.1, 20.5; HRMS m/z (ESI) calcd for C₁₇H₁₄BrN₃O₂ (M+Na⁺) 394.0167, found 394.0159.

2-Amino-4-(3,4-dichlorophenyl)-6,7-dimethyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]pyridine-3-carbonitrile (15), (R═Alk, X═Ar, Q═CN, Y=no ring): 98%; ¹H NMR (DMSO-d₆) δ7.54-7.49 (m, 1H), 7.38 (s, 1H), 7.18-7.16 (m, 1H), 6.83 (s, 2H), 6.03 (s, 1H), 4.43 (s, 1H) 3.35 (s, 3H), 2.35 (s, 3H); ¹³C NMR (DMSO-d₆) δ 161.6, 159.8, 155.5, 148.7, 146.4, 131.3, 130.9, 130.0, 129.8, 128.3, 119.8, 105.1, 97.3, 58.0, 36.9, 31.1, 20.4; HRMS m/z (ESI) calcd for C₁₇H₁₃Cl₂N₃O₂ (M+Na⁺) 384.0283, found 384.0282.

2-Amino-6,7-dimethyl-5-oxo-4-(3,4,5-trimethoxyphenyl)-5,6-dihydro-4H-pyrano[3,2-c]pyridine-3-carbonitrile (16), (R═Alk, X═Ar, Q═CN, Y=no ring): 97%; ¹H NMR (DMSO-d₆) δ 7.01 (s, 2H), 6.42 (s, 2H), 6.06 (s, 1H), 4.35 (s, 1H), 3.69 (s, 6H), 3.60 (s, 3H), 3.32 (s, 3H), 2.34 (s, 3H); ¹³C NMR (DMSO-d₆) δ 161.6, 159.8, 155.4, 153.3, 148.2, 140.9, 137.3, 120.2, 106.2, 105.6, 97.3, 60.6, 58.5, 56.4, 37.5, 31.0, 20.7; HRMS m/z (ESI) calcd for C₂₀H₂₁BrN₃O₅ (M+Na⁺) 406.1379, found 406.1379.

2-Amino-4-(4-isopropylphenyl)-6,7-dimethyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]pyridine-3-carbonitrile (17), (R═Alk, X═Ar, Q═CN, Y=no ring): 81%; ¹H NMR (DMSO-d₆) δ7.15-7.08 (m, 4H), 6.75 (s, 2H), 6.02 (s, 1H), 4.34 (s, 1H), 3.32 (s, 3H), 2.88-2.79 (m, 1H), 2.34 (s, 3H), 1.18 (d, J=6.9 Hz, 6H); ¹³C NMR (DMSO-d₆) δ 161.7, 159.8, 155.4, 148.0, 147.0, 142.7, 127.8, 126.5, 120.2, 106.6, 97.4, 59.3, 37.2, 33.5, 31.1, 24.3, 20.6; HRMS m/z (ESI) calcd for C₂₀H₂₁N₃O₂ (M+Na⁺) 358.1531, found 358.1519.

2-Amino-6,7-dimethyl-4-(3-nitrophenyl)-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]pyridine-3-carbonitrile (18), (R═Alk, X═Ar, Q═CN, Y=no ring): 97%; ¹H NMR (DMSO-d₆) δ8.04 (d, J=7.7 Hz, 1H), 7.95 (s, 1H), 7.65-7.53 (m, 2H) 6.15 (s, 2H), 6.06 (s, 1H), 4.52 (s, 1H), 3.27 (s, 3H), 2.32 (s, 3H); ¹³C NMR (DMSO-d₆) δ 161.8, 159.9, 155.6, 149.1, 148.4, 147.6, 134.9, 130.3, 122.6, 122.1, 120.1, 105.0, 97.5, 56.6, 37.4, 31.1, 20.5; HRMS m/z (ESI) calcd for C₁₇H₁₄N₄O₄ (M+Na⁺) 361.0913, found 361.0909.

2-Amino-4-(3-bromophenyl)-7-methyl-5-oxo-4H,5H-pyrano[4,3-b]pyran-3-carbonitrile (19), (R═Alk, X═Ar, Q═CN, Y=no ring): 87%; ¹H NMR (DMSO-d₆) δ7.43 (d, J=8.0 Hz, 1H), 7.38 (s, 1H), 7.32-7.27 (m, 1H), 7.21 (d, J=8.0 Hz, 1H), 7.15 (s, 2H), 6.26 (s, 1H), 4.34 (s, 1H), 2.22 (s, 3H); ¹³C NMR (DMSO-d₆) δ 163.7, 161.9, 159.1, 158.8, 146.8, 131.3, 130.8, 130.5, 127.3, 122.1, 119.5, 100.6, 98.5, 58.1, 36.6, 19.7; HRMS m/z (ESI) calcd for C₁₆H₁₁BrN₂O₃ (M+Na⁺) 380.9845, found 380.9839.

2-Amino-6-(3,4-dimethoxyphenethyl)-7-methyl-5-oxo-4-(3,4,5-trimethoxyphenyl)-5,6-dihydro-4H-pyrano[3,2-c]pyridine-3-carbonitrile (20),

X═Ar, Q═CN, Y=no ring): 80%; ¹H NMR (DMSO-d₆) δ 6.90 (s, 2H), 6.83 (d, J=8.5 Hz, 1H), 6.69-6.63 (m, 2H), 6.52 (s, 2H), 5.89 (s, 1H), 4.41 (s, 1H), 4.13-3.98 (m, 2H), 3.74 (s, 6H), 3.71 (s, 3H), 3.67 (s, 3H), 3.65 (s, 3H), 2.86-2.70 (m, 2H), 2.19 (s, 3H); ¹³C NMR (DMSO-d₆) δ 161.6, 160.0, 155.6, 153.4, 149.5, 148.4, 147.6, 141.0, 137.4, 131.4, 121.3, 120.5, 113.6, 112.9, 106.6, 105.6, 97.6, 60.5, 58.4, 56.5, 56.3, 56.1, 40.1, 37.2, 33.3, 20.0; HRMS m/z (ESI) calcd for C₂₉H₃₁ N₃O₇ (M+Na⁺) 556.2060, found 556.2057.

2-Amino-6-methyl-5-oxo-4-phenyl-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (21), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 78%; ¹H NMR (DMSO-d₆) δ8.06 (d, J=7.6 Hz, 1H), 7.70-7.66 (m, 1H), 7.52 (d, J=7.6 Hz, 1H), 7.40-7.18 (m, 6H), 6.98 (s, 2H), 4.56 (s, 1H), 3.55 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.5, 159.6, 150.9, 144.9, 139.4, 132.1, 128.8, 128.0, 127.2, 122.9, 122.8, 122.6, 119.9, 115.3, 113.4, 109.8, 59.2, 37.8, 29.8; HRMS m/z (ESI) calcd for C₂₀H₁₅N₃O₂ (M+Na⁺) 352.1062, found 352.1058.

2-Amino-6-methyl-5-oxo-4-(3,4,5-trimethoxyphenyl)-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (22), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 86%; ¹H NMR (DMSO-d₆) δ8.03 (d, J=8.0 Hz, 1H), 7.72-7.66 (m, 1H), 7.55 (d, J=7.6 Hz, 1H), 7.40-7.35 (m, 1H), 7.14 (s, 2H), 6.51 (s, 2H), 4.53 (s, 1H), 3.71 (s, 6H), 3.64 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.6, 159.6, 153.4, 150.8, 140.4, 139.3, 137.5, 132.2, 122.9, 122.7, 120.1, 115.4, 113.3, 109.4, 105.9, 60.5, 58.6, 56.6, 38.2, 29.9; HRMS m/z (ESI) calcd for C₂₃H₂₁N₃O₅ (M+Na⁺) 442.1379, found 442.1389.

2-Amino-4-(3-hydroxy-4-methoxyphenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (23), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 82%; ¹H NMR (DMSO-d₆) δ8.90 (s, 1H), 8.00 (d, J=8.0 Hz, 1H), 7.72-7.67 (m, 1H), 7.55 (d, J=7.6 Hz, 1H), 7.41-7.36 (m, 1H), 7.23 (s, 2H), 6.79 (d, J=6.8 Hz, 1H), 6.61 (s, 2H), 4.37 (s, 1H), 3.71 (s, 3H), 3.55 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.3, 159.3, 150.4, 147.1, 146.8, 139.1, 137.6, 132.0, 122.8, 120.4, 118.7, 115.5, 115.1, 113.2, 112.7, 110.0, 58.8, 56.4, 37.2, 29.8; HRMS m/z (ESI) calcd for C₂₁H₁₇N₃O₄ (M+Na⁺) 398.1117, found 398.1123.

2-Amino-4-(4-hydroxy-3-methoxyphenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (24), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 84%; ¹H NMR (DMSO-d₆) δ8.70 (s, 1H), 8.03 (d, J=7.5 Hz, 1H), 7.70-7.65 (m, 1H), 7.53 (d, J=7.6 Hz, 1H), 7.39-7.34 (m, 1H), 7.08 (s, 2H), 6.84 (s, 1H), 6.79 (d, J=8.2 Hz, 1H), 6.60-6.57 (m, 1H), 4.47 (s, 1H), 3.73 (s, 3H), 3.55 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.5, 159.5, 150.4, 147.7, 146.3, 139.3, 136.0, 132.3, 122.7, 120.4, 116.2, 115.2, 113.3, 113.1, 110.0, 59.0, 56.4, 37.1, 29.8; HRMS m/z (ESI) calcd for C₂₁H₁₇N₃O₄ (M+Na⁺) 398.1117, found 398.1119.

2-Amino-4-(4-hydroxy-3-methoxy-5-nitrophenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (25), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 89%; ¹H NMR (DMSO-d₆) δ10.20 (s, 1H),□8.03 (d, J=8.0 Hz, 1H), 7.73-7.67 (m, 1H), 7.55 (d, J=7.6 Hz, 1H), 7.41-7.36 (m, 1H), 7.23 (s, 4H), 4.61 (s, 1H), 3.85 (s, 3H), 3.54 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.5, 159.7, 150.9, 149.9, 142.3, 139.3, 137.4, 135.8, 132.3, 122.7, 120.0, 117.2, 115.4, 114.8, 113.2, 108.7, 57.9, 57.4, 37.4, 29.9; HRMS m/z (ESI) calcd for C₂₁H₁₆N₄O₆ (M+Na⁺) 443.0968, found 443.0966.

2-Amino-6-methyl-4-(3-nitrophenyl)-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (26), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 77%; ¹H NMR (DMSO-d₆) δ8.11-8.04 (m, 3H), 7.75-7.54 (m, 4H), 7.41-7.36 (m, 1H), 7.00 (s, 2H), 4.78 (s, 1H), 3.56 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 160.0, 150.7, 149.9, 139.3, 134.0, 133.8, 132.4, 131.3, 128.4, 124.3, 122.9, 122.7, 119.4, 115.5, 113.1, 108.8, 56.9, 32.6, 29.8; HRMS m/z (ESI) calcd for C₂₀H₁₄N₄O₄ (M+Na⁺) 397.0913, found 397.0917.

2-Amino-6-methyl-4-(2-nitrophenyl)-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (27), (R═Alk, X═Ar, Q=CN, Y=aromatic ring, Z═H): 73%; ¹H NMR (DMSO-d₆) δ8.06-8.03 (m, 1H), 7.86-7.84 (m, 1H), 7.73-7.66 (m, 1H), 7.61-7.51 (m, 2H), 7.45-7.36 (m, 3H), 7.29 (s, 2H), 5.34 (s, 1H), 3.48 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 160.1, 150.8, 150.0, 139.4, 134.0, 133.8, 132.4, 131.3, 128.5, 124.4, 122.8, 119.5, 115.3, 113.2, 108.8, 57.0, 32.6, 29.8; HRMS m/z (ESI) calcd for C₂₀H₁₄N₄O₄ (M+Na⁺) 397.0913, found 397.0910.

2-Amino-6-methyl-5-oxo-4-(4-pyridinyl)-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (28), (R═Alk, X═HetAr, Q═CN, Y=aromatic ring, Z═H): 79%; ¹H NMR (DMSO-d₆) δ8.45 (d, J=8.5 Hz, 2H), 8.03 (d, J=8.0 Hz, 1H), 7.73-7.67 (m, 1H), 7.54 (d, J=7.5 Hz, 1H), 7.41-7.22 (m, 5H), 4.55 (s, 1H), 3.52 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.3, 159.7, 153.1, 151.2, 150.4, 150.2, 139.3, 132.3, 123.4, 122.9, 119.8, 115.3, 113.1, 107.9, 57.0, 37.6, 29.8; HRMS m/z (ESI) calcd for C₁₉H₁₄N₄O₂ (M+Na⁺) 353.1014, found 353.1017.

2-Amino-6-methyl-5-oxo-4-(3-pyridinyl)-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (29), (R═Alk, X═HetAr, Q═CN, Y=aromatic ring, Z═H): 81%; ¹H NMR (DMSO-d₆) δ8.51 (d, J=3.6 Hz, 1H), 8.40 (m, 1H) 8.05 (m, 1H), 7.72-7.52 (m, 3H), 7.38-7.27 (m, 2H), 7.10 (s, 2H), 4.62 (s, 1H), 3.53 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.5, 159.8, 151.2, 149.8, 148.6, 140.1, 139.5, 135.5, 132.2, 124.4, 123.0, 122.6, 119.7, 115.3, 113.3, 108.7, 58.2, 35.7, 29.7; HRMS m/z (ESI) calcd for C₁₉H₁₄N₄O₂ (M+Na⁺) 353.1014, found 453.1018.

2-Amino-4-(2-furyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (30), (R═Alk, X═HetAr, Q═CN, Y=aromatic ring, Z═H): 75%; ¹H NMR (DMSO-d₆) δ8.04 (d, J=8.0 Hz, 1H), 7.73-7.67 (m, 1H), 7.56 (d, J=7.6 Hz, 1H), 7.41-7.34 (m, 2H), 6.93 (s, 2H), 6.32 (s, 1H), 6.14 (d, J=6.1 Hz, 1H), 4.72 (s, 1H), 3.60 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 155.9, 151.5, 142.3, 142.1, 139.4, 132.1, 122.9, 122.5, 119.5, 115.2, 113.4, 110.9, 107.2, 106.2, 56.7, 31.8, 29.9; HRMS m/z (ESI) calcd for C₁₈H₁₃N₃O₃ (M+Na⁺) 342.0855, found 342.0851.

2-Amino-6-methyl-4-(5-methyl-2-furyl)-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (31), (R═Alk, X═HetAr, Q═CN, Y=aromatic ring, Z═H): 78%; ¹H NMR (DMSO-d₆) δ8.03 (d, J=8.0 Hz, 1H), 7.71-7.57 (m, 2H), 7.40-7.35 (m, 1H), 7.01 (s, 2H), 5.99-5.91 (m, 2H), 4.64 (s, 1H), 3.60 (s, 3H), 2.17 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 159.7, 151.5, 151.5, 145.0, 139.5, 132.5, 131.2, 129.3, 128.8, 123.1, 122.8, 119.6, 115.4, 113.1, 108.3, 56.9, 36.4, 29.9; HRMS m/z (ESI) calcd for C₁₉H₁₅N₃O₃ (M+Na⁺) 356.1011, found 356.1022.

2-Amino-4-(2,3-dichlorophenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (32), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 95%; ¹H NMR (DMSO-d₆) δ8.05 (d, J=8.0 Hz, 1H), 7.75-7.70 (m, 1H), 7.55 (d, J=8.8 Hz, 1H), 7.48-7.38 (m, 2H), 7.27-7.14 (m, 4H), 5.15 (s, 1H), 3.52 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 159.6, 151.5, 145.0, 139.5, 132.5, 131.2, 129.3, 128.8, 123.1, 122.8, 122.7, 119.6, 115.6, 115.4, 113.1, 108.3, 56.9, 36.4, 29.9; HRMS m/z (ESI) calcd for C₂₀H₁₃Cl₂N₃O₂ (M+Na⁺) 420.0283, found 420.0284.

2-Amino-4-(2,6-dichlorophenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (33), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 88%; ¹H NMR (DMSO-d₆) δ8.05 (d, J=8.0 Hz, 1H), 7.72-7.67 (m, 1H), 7.55-7.21 (m, 5H), 6.91 (bs, 2H), 5.62 (s, 1H), 3.52 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.1, 139.6, 137.0, 132.4, 132.2, 129.6, 129.4, 122.8, 122.6, 122.4, 119.1, 115.1, 113.1, 106.7, 55.2, 34.6, 29.5; HRMS m/z (ESI) calcd for C₂₀H₁₃Cl₂N₃O₂ (M+Na⁺) 420.0283, found 420.0280.

2-Amino-4-(3,4-dichlorophenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (34), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 97%; ¹H NMR (DMSO-d₆) δ8.03 (d, J=8.0 Hz, 1H), 7.72-7.67 (m, 1H), 7.55-7.19 (m, 7H), 4.58 (s, 1H), 3.52 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 159.5, 150.9, 145.9, 139.4, 132.5, 131.4, 131.1, 130.2, 129.8, 128.5, 123.0, 122.7, 119.7, 115.5, 113.2, 108.4, 57.7, 37.3, 29.8; HRMS m/z (ESI) calcd for C₂₀H₁₃Cl₂N₃O₂ (M+Na⁺) 420.0283, found 420.0274.

2-Amino-4-(3-chlorophenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (35), (R═Alk, X═Ar, Q=CN, Y=aromatic ring, Z═H): 91%; ¹H NMR (DMSO-d₆) δ8.02 (d, J=8.0 Hz, 1H), 7.73-7.68 (m, 1H), 7.58-7.16 (m, 8H), 4.55 (s, 1H), 3.53 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 159.5, 150.8, 147.4, 139.3, 133.4, 131.0, 128.0, 126.8, 122.8, 122.6, 120.1, 113.1, 108.7, 57.8, 37.3, 29.8; HRMS m/z (ESI) calcd for C₂₀H₁₄ClN₃O₂ (M+Na⁺) 386.0673, found 386.0677.

2-Amino-4-(3-fluorophenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (36), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 90%; ¹H NMR (DMSO-d₆) δ8.05-8.02 (m, 1H), 7.72-7.67 (m, 1H), 7.54 (d, J=7.5 Hz, 1H), 7.40-7.00 (m, 7H), 4.58 (s, 1H), 3.54 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 159.6, 150.9, 147.8, 139.4, 132.2, 130.8, 124.0, 122.7, 119.9, 115.5, 114.9, 114.6, 114.2, 113.9, 133.2, 108.9, 58.1, 37.7, 29.9; HRMS m/z (ESI) calcd for C₂₀H₁₄FN₃O₂ (M+Na⁺) 307.0968, found 370.0963.

2-Amino-4-(3-bromophenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (37), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 93%; ¹H NMR (DMSO-d₆) δ8.07 (d, J=8.0 Hz, 1H), 7.73-7.68 (m, 1H), 7.57-7.24 (m, 7H), 6.98 (s, 1H),4.59 (s, 1H), 3.57 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.5, 159.0, 151.8, 147.9, 139.3, 133.8, 131.0, 128.4, 122.8, 120.0, 113.0, 108.7, 105.4, 57.87 37.5, 29.7; HRMS m/z (ESI) calcd for C₂₀H₁₄BrN₃O₂ (M+Na⁺) 430.0167, found 430.0160.

2-Amino-4-(3-bromo-4-fluorophenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (38), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 95%; ¹H NMR (DMSO-d₆) δ8.03 (d, J=8.0 Hz, 1H), 7.72-7.23 (m, 8H), 4.59 (s, 1H), 3.54 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 159.5, 150.9, 143.0, 139.3, 133.0, 132.8, 132.3, 129.5, 123.0, 122.8, 119.9, 117.2, 115.5, 113.2, 108.6, 58.1, 36.7, 29.7; HRMS m/z (ESI) calcd for C₂₀H₁₃BrFN₃O₂ (M+Na⁺) 448.0073, found 448.0056.

2-Amino-4-(3,5-dibromo-4-hydroxyphenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (39), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 92%; ¹H NMR (DMSO-d₆) δ8.03 (d, J=8.0 Hz, 1H), 7.72-7.23 (m, 8H), 4.50 (s, 1H), 3.55 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 159.5, 150.9, 150.2, 139.5, 139.3, 132:2, 131.8, 123.0, 122.8, 119.9, 115.6, 113.4, 112.3, 108.7, 58.0, 37.5, 29.9; HRMS m/z (ESI) calcd for C₂₀H₁₄Br₂N₃O₃ (M+Na⁺) 523.9221, found 523.9210.

2-Amino-4-(3-bromo-4-hydroxy-5-methoxyphenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (40), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 94%; ¹H NMR (DMSO-d₆) δ9.16 (s, 1H), 8.02 (d, J=8.0 Hz, 1H), 7.71-7.53 (m, 2H), 7.39-7.34 (m, 1H), 7.15 (s, 2H), 6.86 (s, 1H), 6.82 (s, 1H), 4.47 (s, 1H), 3.77 (s, 3H), 3.54 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 159.5, 150.6, 148.8, 143.3, 139.2, 136.9, 132.2, 123.5, 123.0, 122.7, 120.1, 115.5, 113.3, 111.8, 109.8, 109.3, 58.5, 57.0, 37.2, 29.9; HRMS m/z (ESI) calcd for C₂₁H₁₆BrN₃O₄ (M+Na⁺) 476.0222, found 476.0226.

2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (41), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 95%; ¹H NMR (DMSO-d₆) δ8.03 (d, J=8.0 Hz, 1H), 7.72-7.67 (m, 1H), 7.55 (d, J=8.0 Hz, 1H), 7.40-7.35 (m, 1H), 7.21 (s, 2H), 6.98 (d, J=1.7 Hz, 1H), 6.92 (d, J=1.7 Hz, 1H) 4.56 (s, 1H), 3.79 (s, 3H), 3.71 (s, 3H), 3.56 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 159.6, 153.8, 150.9, 145.3, 142.2, 139.3, 132.3, 123.3, 123.0, 122.5, 120.1, 117.0, 115.5, 113.3, 113.0, 108.9, 60.5, 58.2, 56.5, 37.4, 29.8; HRMS m/z (ESI) calcd for C₂₂H₁₈BrN₃O₄ (M+Na⁺) 490.0379, found 490.0371.

2-Amino-4-(3,5-dibromophenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (42), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 82%; ¹H NMR (DMSO-d₆) δ 8.05 (d, J=8.3 Hz, 1H), 7.65-7.57 (m, 2H), 7.48-7.38 (m, 4H), 7.29 (s, 2H), 4.60 (s, 1H), 3.56 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.4, 159.7, 151.2, 149.6, 132.6, 130.1, 122.8, 115.5, 113.2, 107.8, 57.5, 37.6, 29.7; HRMS m/z (ESI) calcd for C₂₀H₁₃Br₂N₃O₂ (M+Na⁺) 507.9272, found 507.9257.

2-Amino-4-(3-bromo-4-methoxyphenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (43), (R═Alk, X═Ar, Q═CN, Y=aromatic ring, Z═H): 64%; ¹H NMR (DMSO-d₆) δ8.02 (d, J=8.0 Hz, 1H), 7.72-7.67 (m, 1H), 7.57-7.54 (d, J=7.5 Hz, 1H), 7.38 (s, 2H), 7.22 (s, 3H), 7.01 (d, J=7.0 Hz, 1H), 4.05 (s, 1H), 3.79 (s, 3H), 3.54 (s, 3H); ¹³C NMR (DMSO-d₆) δ 160.3, 159.3, 154.7, 150.5, 139.1, 138.6, 132.4, 128.7, 122.8, 122.6, 120.2, 115.6, 113.1, 110.7, 109.1, 58.1, 56.8, 37.0, 29.8; HRMS m/z (ESI) calcd for C₂₁H₁₆BrN₃O₃ (M+Na⁺) 460.0273, found 460.0287.

TABLE 1 Antiproliferative activity of pyrano-[3,2-c]-pyridones and related heterocycles. cell viability^(a) GI₅₀, μM analogue HeLa MCF-7 3

0.013 ± 0.003 0.015 ± 0.008 4

0.18 ± 0.02 0.025 ± 0.06  8

0.33 ± 0.06 Not tested 9

0.58 ± 0.14 Not tested 10

1.1 ± 0.8 Not tested 11

2.7 ± 1.1 Not tested 12

3.5 ± 1.3 Not tested 13

6.4 ± 1.1 Not tested 14

6.5 ± 1.3 Not tested 15

18.3 ±  2.9 Not tested 17

>100 Not tested 20

22.7 ±  6.4 Not tested 22

0.24 ± 0.02 1.0 ± 0.3 25

0.63 ± 0.02 0.71 ± 0.12 37

0.74 ± 0.03 0.003 ± 0.001 38

0.27 ± 0.03 0.81 ± 0.08 39

0.27 ± 0.02 0.43 ± 0.01 40

0.047 ± 0.010 0.39 ± 0.16 41

0.014 ± 0.003 0.38 ± 0.03 42

0.077 ± 0.006 0.075 ± 0.007 43

0.41 ± 0.04 0.5 ± 0.1 ^(a)Concentration required to reduce the viability of cells by 50% after 48 h of treatment with indicated compounds relative to DMSO control ± SD from two independent experiments, each performed in 8 replicates, determined by MTT assay.

Since many clinically used anticancer agents induce apoptosis in cancer cells, we tested the pyranopyridone analogues for their ability to induce apoptosis in Jurkat (model for human T-cell leukemia) cells using the flow cytometric annexin-V/propidium iodide assay (FIG. 1). Compounds 8-14, exhibiting submicromolar or low micromolar potencies for the inhibition of proliferation of HeLa cells, were found to be strong inducers of apoptosis in Jurkat cells at 5 μM concentrations. The magnitude of apoptosis induction is comparable to the known antimitotic agent colchicine used at the same concentration. In contrast, compounds 15, 17, 20, which are much less potent or totally inactive in the HeLa MTT assay, do not induce apoptosis in Jurkat cells at this concentration.

FIG. 1 illustrates induction of apoptosis in Jurkat cells treated for 36 h with DMSO control, colchicine (5 μM) and selected pyridone library analogues (5 μM) in the flow cytometric annexin-V/propidium iodide assay. Error bars represent data

Since endonuclease-mediated cleavage of nuclear DNA resulting in formation of oligonucleosomal DNA fragments (180-200 base pairs long) is a hallmark of apoptosis in many cell types, apoptosis was further investigated with the DNA laddering assay (FIG. 2). Jurkat cells were treated with DMSO (lane A), analogues 40 and 41 (lanes B and C), and paclitaxel (lane D) for 36 h. After that the cellular DNA was isolated and electrophoresed in a 1.5% agarose gel. The characteristic ladder pattern was obtained with compounds 40, 41 and paclitaxel. No laddering was observed when the cells were treated with DMSO control.

FIG. 2 illustrates DNA laddering in Jurkat cells after 36 h of treatment. (A) DMSO control, (B) 40 at 1.5 μM, (C) 41 at 1.5 μM, (D) paclitaxel at 0.4 μM, and (E) Molecular weight marker (KB).

Morphological changes of cells treated with potent analogues can be visually observed with light microscopy (FIG. 3). The phenotypic changes of Jurkat cells, such as formation of finger-like extensions and shriveling, become apparent as early as 2 h after their treatment with 40 (C), and 41 (D).

FIG. 3 illustrates light microscopy pictures. Treatment of Jurkat cells with 40 (C) and 41 (D) for 2 hours induces formation of finger-like extensions (black arrows) and shriveling (white arrows). 0.1% DMSO (A) and a known anticancer agent (B) were used as controls. All compounds are used at 1 μM. The scale bar indicates 40 μm.

Our libraries of compounds exert their antiproliferative properties through inhibition of tubulin dynamics, thereby inducing mitotic arrest and initiating apoptosis in cancer cells. Indeed, the flow cytometric cell cycle analysis, performed with pyranoquinolones 40 and 41 using the Jurkat cell line, shows a pronounced cell cycle arrest in the G2/M phase (Table 2). This effect is characteristic of antimitotic agents disrupting microtubule assembly.

TABLE 2 Flow cytometric cell cycle analysis of Jurkat cells. % relative DNA content^(a) compound G0/G1 S G2/M DMSO 56 ± 2 21 ± 3 20 ± 2 40 27 ± 3 22 ± 2 47 ± 3 41 20 ± 2 28 ± 2 49 ± 2 ^(a)% Relative DNA content ± SD after 24 h treatment of Jurkat cells with indicated compounds from two independent experiments each performed in triplicate. Compounds 40, 41 are used at 1 μM, obtained using the flow cytometric Vybrant Orange staining assay. The remaining % DNA content is found in sub G0/G1 region.

To obtain further support for the proposed antitubulin mechanism of action for our heterocycles, we assessed the effect of pyranoquinolones on in vitro tubulin polymerization. In this assay microtubule formation is monitored by the increase in fluorescence intensity of the reaction mixture. Paclitaxel exhibited potent enhancement of microtubule formation relative to the effect of DMSO control (FIG. 4). In contrast, library members 40 and 41 displayed potent microtubule destabilizing effect in a manner similar to the known tubulin polymerization inhibitor podophyllotoxin.

FIG. 4 illustrates the effect of our compounds on in vitro tubulin polymerization. Paclitaxel (3 μM, square markers) promotes microtubule formation relative to 0.05% DMSO control (triangle markers). 40 (25 μM, dash markers), 41 (25 μM, circle markers) and podophyllotoxin (25 μM, cross markers) completely suppress tubulin polymerization. Each data point is a mean of two independent experiments producing similar results. 

1. A method of treating a disorder responsive to the induction of apoptosis or antiproliferation or vascular disruption in an animal suffering therefrom, wherein the disorder is a cancer selected from the group consisting of cervical carcinoma, breast carcinoma, acute lymphatic leukemia, chronic lymphocytic leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, and hairy cell leukemia, comprising administering to an animal in need of such treatment an effective amount of a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: R₁ is hydrogen, haloalkyl, aryl, fused aryl, carbocyclic, a heterocyclic group, a heteroaryl group, C₁₋₁₀ alkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, carbocycloalkyl, heterocycloalkyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitroalkyl, cyanoalkyl, acetamidoalkyl, acyloxyalkyl, hydroxyl, alkoxy, acetoxy, amino, alkylamino, acetamido; X is optionally substituted and is aryl, heteroaryl, saturated carbocyiclic, partially saturated carbocyclic, saturated heterocyclic, partially saturated heterocyclic, arylalkyl, heteroarylalkyl; Q is CN, COR₂, CO₂R₂, CONR_(x)R_(y), CSNR_(x)R_(y) or SO₂R₂, wherein R₂, R_(x) and R_(y) are independently hydrogen, haloalkyl, aryl, fused aryl, carbocyclic, a heterocyclic group, a heteroaryl group, C₁₋₁₀ alkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, carbocycloalkyl, heterocycloalkyl, hydroxyalkyl, aminoalky, carboxyalkyl, nitroalkyl, cyanoalkyl, acetamidoalkyl, acyloxyalkyl; or R_(x) and R_(y) are taken together with the nitrogen to which they are attached to form a heterocycle; P is hydrogen, halogen, NR₃R₄, NHCOR₃, N(COR₃)₂, N(COR₃)(COR₄), NHCONR₃R₄, NHCSNR₃R₄, N═CHOR₃, N═CHR₃, wherein R₃ and R₄ are independently hydrogen, haloalkyl, C₁₋₄ alkyl, aryl, heteroaryl, or R₃ and R₄ are combined together with the group attached to them to form a heterocycle; Y is no ring, aromatic ring, saturated carbocyclic ring, partially saturated carbocyclic ring, saturated heterocyclic ring, partially saturated heterocyclic ring; Z is hydrogen, halogen, haloalkyl, C₁₋₁₀ alkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, nitroalkyl, cyanoalkyl, acetamidoalkyl, acyloxyalkyl, hydroxy, alkoxy, acetoxy, amino, alkylamino, acetamido, cyano, nitro, carobxy, thiol, azido, methylendioxy, carbonylamido, or alkysulfanyl. 